Nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) have been exploited successfully in combinatorial biosynthesis of "unnatural" natural products for drug lead discovery and optimization. Hybrid NRPS-PKS systems will allow the production of novel metabolites by incorporating both amino acids and carboxylic acids, greatly expanding the size and structural diversity of resulting combinatorial biosynthetic libraries. A great challenge in constructing hybrid NRPS-PKS systems lies in the revelation of the molecular basis for intermodular communication between NRPS and PKS. I propose to continue a study of the biochemistry and genetics of the production of the bleomycins (BLMs) and related metabolites as a model for hybrid peptide-polyketide natural product biosynthesis. My long-term goal is to construct hybrid NRPS-PKS systems to produce novel cell-permeable bioactive molecules and pharmacological leads. My short-term goals for this project are (1) to biochemically and genetically characterize the BLM biosynthetic pathway and (2) to make novel BLM congeners through combinatorial biosynthesis for the discovery and development of clinically useful anticancer drugs. My hypotheses are: (1) the BLM megasynthetase is a "natural" hybrid NRPS-PKS system, the studies of which will reveal how nature integrates NRPS and PKS proteins into a hybrid NRPS-PKS system; (2) the initiation and termination of BLM biosynthesis and the formation of the bithiazole moiety of BLM are unprecedented in peptide biosynthesis, the characterization of which will uncover novel chemistry for NRPSs; (3) the BLM megasynthetase provides a novel platform for combinatorial biosynthesis to make clinically valuable anticancer drugs. My specific aims are: (1) functional analysis of the BLM biosynthetic gene cluster in vivo; (2) biochemical characterization of the BLM megasynthetase in vitro; (3) production of novel BLMs by engineering BLM biosynthesis and evaluation of them as anticancer drugs. The outcomes of these studies will (1) expand the repertoire of NRPS and PKS genes for combinatorial biosynthesis, (2) lay the foundation for rational construction of hybrid NRPS-PKS systems, and (3) potentially lead to the production of novel BLMs with improved therapeutic efficacy as anticancer drugs.